Dose-dependent tazepam modulation of amplitude-temporal characteristics of thalamocortical responses and the constant potential of the sensorimotor cortex in rabbits at eye opening

The pronounced benzodiazepine (antiphobic) modulation of the amplitude-temporal parameters of different components of the thalamocortical responses (TCR) of the sensorimotor cortex is observed in rabbits in their early postnatal ontogeny. This modulation is of a dose-dependent character and is registered not after the injection of tazepam in a concentration of the "therapeutic tranquilizing window" but also in the psychotoxic plasma range. A gradual increase in blood tazepam concentration in a young rabbit pup is accompanied by the wave-like and differential decrease in the amplitude of the second and third positive (P2 and P3) and third negative (N3) TCR components, while the second negative (N2) and fourth positive (P4) components tend to a wave-like increase. The dose-dependent dynamics of tazepam modulation of the P2, P3, and N3 latencies is characterized by a wave-like and differential increase. The latency of P4 decreases slightly and that of the N2 increases with a low degree of significance. The selective dynamics of benzodiazepine modulation appears to be related with peculiarities of the electrogenesis of each of the components. The dose-dependent modulation of the level of cortical DC potential is of the same character as the respective amplitude changes in P2, P3, and N3, but its fluctiatuons are more pronounced.     

Role of Zn in epigallocatechin gallate affecting the growth of PC-3 cells

Green tea has chemo-preventive effects to human carcinoma including prostate cancer. Epigallocatechin gallate (EGCG) is the major active component in green tea. Zn(2+) is indispensable to our health, and plays an important role in the normal function and pathology of the prostate gland, and might be a good marker for diagnosing prostate cancer. Effects of Zn(2+), EGCG and their interactions on the growth of androgen-insensitive prostate cancer cell (PC-3) were investigated in the present paper. The results show that Zn(2+) and EGCG inhibited the growth of PC-3 cells in a time- and dose-dependent manner, but effects of interactions of EGCG with Zn(2+) were extremely dependent on their concentrations and added orders. Inhibitory effects of Zn(2+) were significantly decreased in the presence of EGCG on PC-3 cell growth. Therefore, we hypothesize that complexation of EGCG with Zn(2+) might be responsible for the observed decrease of the bioactivities of Zn(2+) against PC-3 cells.     

ATP-dependent aggregation of single-stranded DNA by a bacterial SMC homodimer.

SMC (structural maintenance of chromosomes) proteins are putative ATPases that are highly conserved among Bacteria, Archaea and Eucarya. Eukaryotic SMC proteins are implicated in a diverse range of chromosome dynamics including chromosome condensation, dosage compensation and recombinational repair. In eukaryotes, two different SMC proteins form a heterodimer, which in turn acts as the core component of a large protein complex. Despite recent progress, no ATP-dependent activity has been found in individual SMC subunits. We report here the first biochemical characterization of a bacterial SMC protein from Bacillus subtilis. Unlike eukaryotic versions, the B.subtilis SMC protein (BsSMC) is a simple homodimer with no associated subunits. It binds preferentially to single-stranded DNA (ssDNA) and has a ssDNA-stimulated ATPase activity. In the presence of ATP, BsSMC forms large nucleoprotein aggregates in a ssDNA-specific manner. Proteolytic cleavage of BsSMC is changed upon binding to ATP and ssDNA. The energy-dependent aggregation of ssDNA might represent a primitive type of chromosome condensation that occurs during segregation of bacterial chromosomes.     

Identification of a novel non-structural maintenance of chromosomes (SMC) component of the SMC5-SMC6 complex involved in DNA repair

Structural maintenance of chromosomes (SMC) proteins play central roles in chromosome organization and dynamics. They have been classified into six subtypes, termed SMC1 to SMC6, and function as heterodimer components of large protein complexes that also include several non-SMC proteins. The SMC2-SMC4 and SMC1-SMC3 complexes are also known as condensin and cohesin, respectively, but the recently identified SMC5 and SMC6 complex is less well characterized. Here, we report that NSE1 from Saccharomyces cerevisiae encodes a novel non-SMC component of the SMC5(Yol034wp)-SMC6(Rhc18p) complex corresponding to the 2-3-MDa molecular mass. Nse1p is essential for cell proliferation and localizes primarily in the nucleus. nse1 mutants are highly sensitive to DNA-damaging treatments and exhibit abnormal cellular morphologies, suggesting aberrant mitosis as a terminal morphological phenotype. These results are consistent with the reported features of the Schizosaccharomyces pombe SMC6 gene, rad18, which is thought to be involved in recombinational DNA repair. We conclude that Nse1p and the SMC5-SMC6 heterodimer together form a high molecular mass complex that is conserved in eukaryotes and required for both DNA repair and proliferation.     

真核生物SMC基因家族中拷贝数目的长期稳定进化

Members of the Structural Maintenance of Chromosome (SMC) family have long been of interest to molecular and evolutionary biologists for their role in chromosome structural dynamics, particularly sister chromatid cohesion, condensation, and DNA repair. SMC and related proteins are found in all major groups of living organisms and share a common structure of conserved N and C globular domains separated from the conserved hinge domain by long coiled-coil regions. In eukaryotes there are six paralogous proteins that form three heterodimeric pairs, whereas in prokaryotes there is only one SMC protein that homodimerizes. From recently completed genome sequences, we have identified SMC genes from 34 eukaryotes that have not been described in previous reports. Our phylogenetic analysis of these and previously identified SMC genes supports an origin for the vertebrate meiotic SMC1 in the most recent common ancestor since the divergence from invertebrate animals. Additionally, we have identified duplicate copies due to segmental duplications for some of the SMC paralogs in plants and yeast, mainly SMC2 and SMC6, and detected evidence that duplicates of other paralogs were lost, suggesting differential evolution for these genes. Our analysis indicates that the SMC paralogs have been stably maintained at very low copy numbers, even after segmental (genome-wide) duplications. It is possible that such low copy numbers might be selected during eukaryotic evolution, although other possibilities are not ruled out.     

Structure and DNA-binding activity of the Pyrococcus furiosus SMC protein hinge domain

Structural Maintenance of Chromosomes (SMC) proteins are essential for a wide range of processes including chromosome structure and dynamics, gene regulation, and DNA repair. While bacteria and archaea have one SMC protein that forms a homodimer, eukaryotes possess three distinct SMC complexes, consisting of heterodimeric pairs of six different SMC proteins. SMC holocomplexes additionally contain several specific regulatory subunits. The bacterial SMC complex is required for chromosome condensation and segregation. In eukaryotes, this function is carried out by the condensin (SMC2-SMC4) complex. SMC proteins consist of N-terminal and C-terminal domains that fold back onto each other to create an ATPase "head" domain, connected to a central "hinge" domain via a long coiled-coil region. The hinge domain mediates dimerization of SMC proteins and binds DNA. This activity implicates a direct involvement of the hinge domain in the action of SMC proteins on DNA. We studied the SMC hinge domain from the thermophilic archaeon Pyrococcus furiosus. Its crystal structure shows that the SMC hinge domain fold is largely conserved between archaea and bacteria as well as eukarya. Like the eukaryotic condensin hinge domain, the P. furiosus SMC hinge domain preferentially binds single-stranded DNA (ssDNA), but its affinity for DNA is weaker than that of its eukaryotic counterpart, and point mutations reveal that its DNA-binding surface is more confined. The ssDNA-binding activity of its hinge domain might play a role in the DNA-loading process of the prokaryotic SMC complex during replication.     

SMC Proteins,New Players in the Maintenance of Genomic Stability

Homologous recombination (HR) is one of the key mechanisms responsible for the repair of DNA double-strand breaks (DSBs), including those that occur during DNA replication. Recent studies in yeast and mammals have uncovered that the SMC complexes cohesins and Smc5-Smc6 are recruited to induced DSBs, and play a role in the maintenance of genome stability by favouring SCR as the main recombinational DSB repair mechanism. These new results raise intriguing questions such as whether SMC proteins might play a functional role at collapsed replication forks, which may represent the main source of spontaneous recombinogenic damage. A deeper knowledge of the role of SMC proteins in DSB repair should contribute to a better understanding of chromosome dynamics and stability.     

Age-related dynamics of thalamic-cortical evoked potentials in the rabbit in the early period of life

Data have been obtained on development of evoked potentials in the sensorimotor cortex to electrical stimulation of the thalamic ventroposterolateral nucleus (VPL) in rabbits in early ontogeny. In 3-5 days rabbits, under four times increase of threshold electric stimulation of VPL the thalamocortical response (TCR) is presented by a positive-negative potential with a long latency and minimum amplitude parameters. Second and third TCR positive components to increasing of threshold value of electric stimulation 4 times, are differentiated from 7-8 days age. Age dynamics of TCR amplitude-temporal parameters is characterized by a shortening of latency and an increase of oscillations amplitude, most expressed at 2-3 weeks of postnatal life. TCR of one month rabbit to increased threshold electrical stimulation of VPL is presented by short-latency positive-negative oscillation with a positive phase consisting of three components with successively increasing amplitudes.     

Mutations in cohesin complex members SMC3 and SMC1A cause a mild variant of cornelia de Lange syndrome with predominant mental retardation

Mutations in the cohesin regulators NIPBL and ESCO2 are causative of the Cornelia de Lange syndrome (CdLS) and Roberts or SC phocomelia syndrome, respectively. Recently, mutations in the cohesin complex structural component SMC1A have been identified in two probands with features of CdLS. Here, we report the identification of a mutation in the gene encoding the complementary subunit of the cohesin heterodimer, SMC3, and 14 additional SMC1A mutations. All mutations are predicted to retain an open reading frame, and no truncating mutations were identified. Structural analysis of the mutant SMC3 and SMC1A proteins indicate that all are likely to produce functional cohesin complexes, but we posit that they may alter their chromosome binding dynamics. Our data indicate that SMC3 and SMC1A mutations (1) contribute to approximately 5% of cases of CdLS, (2) result in a consistently mild phenotype with absence of major structural anomalies typically associated with CdLS, and (3) in some instances, result in a phenotype that approaches that of apparently nonsyndromic mental retardation.     

Kinetics and Energetics of Mg2+,ATP-Dependent Ca2+ Transport in the Plasma Membrane of Smooth Muscle Cells

Calcium ions play a crucial role in the excitation/contraction coupling in smooth muscles. I would like to interpret the biochemical mechanisms underlying Ca²⁺ exchange and dynamics of such an exchange in the smooth muscles. Particular emphasis is laid on the examination of kinetic, energetic, and catalytic properties of the membrane-linked energy-dependent Ca²⁺-transporting systems involved in regulation of the intracellular Ca²⁺ concentration in smooth muscle cells (SMC). It was suggested that the Mg²⁺,ATP-dependent plasma membrane calcium pump (Ca²⁺,Mg²⁺-ATPase) plays a key role in regulation of the Ca²⁺ concentration in SMC. The purpose of this review is to analyze some of our own results concerning kinetic, energetic, and catalytic properties of the calcium pump of the SMC plasma membrane. In our experiments, we used different biochemical models (namely, fractions of the membrane subcellular structures, highly purified Ca²⁺,Mg²⁺-ATPase of the SMC plasma membrane solubilized and reconstituted in the lyposomes, and suspension of digitonin-treated SMC) and a number of methods (including preparative biochemistry, enzymology, membranology, tracer ⁴⁵Ca²⁺ flux analysis, and chemical and enzymological kinetics). We have shown that sodium azide-insensitive Mg²⁺,ATP-dependent Ca²⁺ accumulation in ureter smooth muscle microsomes is determined by two components. One component represents the Mg²⁺,ATP-dependent calcium pump of the sarcoplasmic reticulum functionally potentiated by Ca²⁺-precipitating permeating anions, oxalate or phosphate and inhibited by thapsigargin or cyclopiazonic acid, the highly selective inhibitors of the calcium pump of sarco(endo)plasmic rerticulum. Another component represents the Mg²⁺,ATP-dependent calcium pump of the plasma membrane functionally potentiated by phosphate. This pump is not inhibited by thapsigargin and cyclopiazonic acid. The effects of temperature, dielectric permeability (D), and ionic strength on the activity of purified Ca²⁺,Mg²⁺-ATPase solubilized from the myometrial sarcolemma were studied. The results suggest that changes in the polarity of the incubation medium markedly affect the activity of transport Ca²⁺,Mg²⁺-ATPase, and electrostatic interactions between the enzyme activity center and specific ligands (Mg·ADP^-, in particular) significantly contribute to the energetics of ATP hydrolysis. Therefore, our data show that changes in the incubation medium polarity significantly affects the ATP-hydrolase activity of Ca²⁺,Mg²⁺-ATPase solubilized from the SMC plasma membranes, and electrostatic interactions between the enzyme active sites and reactants (in particular, Mg·ADP^-) contribute to a significant extent to the energetics of ATP hydrolysis. We cannot rule out that under physiological conditions the local D values of the myoplasm may differ from that of water, and, moreover, may change (especially near the membrane surface) depending on the metabolic level of SMC. We suppose that local changes in the cytoplasmic D value will affect the plasma membrane calcium pump and, consequently, the efficiency of control of intracellular Ca²⁺ homeostasis in smooth muscle. So, our biochemical models are suitable experimental objects for studying the kinetic, energetic, and catalytic properties of the Mg²⁺,ATP-dependent calcium pump of the SMC plasma membrane. In addition, our data might be useful for screening of the mechanisms underlying the action of different physico-chemical factors involved in modulation of the contraction/relaxation cycle.     

Effects of Some Organic Cations on Generator Potential of Crayfish Stretch Receptor

The generator potential of both slowly and rapidly adapting crayfish stretch receptor cells can still be elicited by mechanical stimuli when all the Na of the bathing medium is replaced by various organic cations. In the presence of tris(hydroxymethyl)aminomethane (Tris), the generator potential is particularly large, about 30–50 % of that in the control saline, while spike electrogenesis of the cell is abolished. Persistence of the generator response is not due to retention of Na by a diffusion barrier, and ionic contributions to the electrogenesis by Ca and Cl can also be excluded. Thus, whereas the electrogenesis of the generator membrane must be due to an increased permeability to monovalent cations, the active receptor membrane appears to be less selective for different monovalent cations than is the receptor component of some other cells, or the conductile component of the stretch receptor neuron.     

Recruitment of the actin-binding protein HIP-55 to the immunological synapse regulates T cell receptor signaling and endocytosis

Actin cytoskeleton dynamics critically regulate T cell activation. We found that the cytoplasmic adaptor HIP-55, a Src/Syk-kinases substrate and member of the drebrin/Abp1 family of actin-binding proteins, localized to the T cell-antigen-presenting cell (APC) contact site in an antigen-dependent manner. Using green fluorescent protein fusion proteins, both Src homology 3 (SH3) and actin binding domains were found necessary for recruitment at the T cell-APC interface. HIP-55 was not implicated in conjugate formation and actin polymerization but regulated distal signaling events through binding and activation of hematopoietic progenitor kinase 1 (HPK1), a germinal center kinase (GCK) family kinase involved in negative signaling in T cells. Using RNA interference and overexpression experiments, the HIP-55-HPK1 complex was found to negatively regulate nuclear factor of activated T cell (NFAT) activation by the T cell antigen receptor. Moreover, we show that HIP-55, which partly co-localized with early endocytic compartments, promoted both basal and ligand-dependent T cell receptor (TCR) down-modulation, resulting in a decreased TCR expression. SH3 and actin-depolymerizing factor homology domains were required for this function. As controls, the expression of CD28 and the glycosylphosphatidylinositol-linked protein CD59 was not affected by HIP-55 overexpression. These results suggest that, in addition to binding to HPK1, HIP-55 might negatively regulate TCR signaling through down-regulation of TCR expression. Our findings show that HIP-55 is a key novel component of the immunological synapse that modulates T cell activation by connecting actin cytoskeleton and TCRs to gene activation and endocytic processes.     

Effect of extirpation of the sensorimotor cortex on the formation of conditioned reflexes to a complex signal in the ontogeny of the cat

In kittens of the age from 39 days to 4 months the influence was studied of the sensorimotor cortex (SMC) ablation on formation and preservation of previously elaborated reflex to a simultaneous complex stimulus (light + sound) with extinction of reaction to nonreinforced components of the complex signal. It is shown that SMC ablation in the age up to 2.5 months does not affect the formation or preservation of the conditioned reflex. The SMC ablation in preliminarily trained kittens in the age of 3.5 months and older leads to disinhibition of components differentiation with preservation of the positive conditioned reflex to the complex signal. In untrained kittens of the same age the SMC ablation leads to disability of animals to inhibit the motor reaction to differentiation signal. The question is discussed of SMC significance in the ontogenesis at formation of adequate forms of behaviour requiring heterosensory interaction.     

The Action of Tetrodotoxin on Electrogenic Components of Squid Giant Axons

Voltage clamp measurements on squid giant axons show that externally applied puffer fish poison, tetrodotoxin, eliminates only the initial inward current component of spike electrogenesis and does not affect the subsequent outward current. The selective effect on Na activation, which is reversible, confirms the view that the movements of Na and K during spike electrogenesis occur at structurally different sites on the membrane. Spike electrogenesis is also blocked when tetrodotoxin is injected into the axon, but the interior of the membrane appears to be somewhat less sensitive to the poison. Differences in reactivity of various electrogenic membrane components to tetrodotoxin are discussed as signifying differences in chemical structures.     

Asymmetry of the transcallosal responses in the parietal cortex of kittens in the 1st month of life

In acute experiments on kittens the process of formation of asymmetry of transcallosal responses (TCR) was studied in multiple leads from symmetrical points of the parietal cortex. By the early positive-negative TCR complex, vanishing as a result of callosotomy, predominance of positive components in the right hemisphere was found in 2-7 days kittens, whereas in 8-24 days animals the left hemisphere dominated by both phases of responses. By the late TCR component preserved after section of the callosal body, left-hemispheric asymmetry was found in the elder group of kittens; it was absent in the younger animals. TCR asymmetry in the parietal cortex depended on the sex of the animals. With their age its inversion and enhancement took place. This process is based on the increase of TCR amplitude in the left hemisphere, with no increase in the right hemisphere.     

Vimentin binds IRAP and is involved in GLUT4 vesicle trafficking

Cysteine-rich protein 2 (CRP2) is a cofactor for smooth muscle cell (SMC) differentiation. Here, we examined the mechanism of CRP2 distribution dynamics during SMC differentiation. CRP2 protein directly associated with F-actin through its N-terminal LIM domain and Gly-rich region, as determined by ELISA. In undifferentiated cells that contain few actin stress fibers, CRP2 was broadly distributed throughout the whole cell, including the nucleus. After induction of SMC differentiation, CRP2 localized to actin stress fibers as they formed. The stress fiber-localized CRP2 entered the nucleus because of induced actin depolymerization. These CRP2 dynamics were reproduced by in silico simulation. CRP2 localization dynamics, which affect CRP2 function, are regulated by the formation of actin stress fibers in conjunction with SMC differentiation.     

SMC蛋白的结构和功能

近年来新发现的一类蛋白－染色体结构维持蛋白（SMC蛋白,structural maintenance of chromosome proteins)与染色体结构细胞周期性的动态变化紧密相关,它们参与有丝分裂染色体的集缩和分离,性染色体的剂量补偿效应,姐妹染色单体的内聚作用（cohesion),遗传重组和DNA修复等过程,本从生化特性和生物学功能两方面叙述了对SMC蛋白的研究。     

TCR Triggering by pMHC Ligands Tethered on Surfaces via Poly(Ethylene Glycol) Depends on Polymer Length

Antigen recognition by T cells relies on the interaction between T cell receptor (TCR) and peptide-major histocompatibility complex (pMHC) at the interface between the T cell and the antigen presenting cell (APC). The pMHC-TCR interaction is two-dimensional (2D), in that both the ligand and receptor are membrane-anchored and their movement is limited to 2D diffusion. The 2D nature of the interaction is critical for the ability of pMHC ligands to trigger TCR. The exact properties of the 2D pMHC-TCR interaction that enable TCR triggering, however, are not fully understood. Here, we altered the 2D pMHC-TCR interaction by tethering pMHC ligands to a rigid plastic surface with flexible poly(ethylene glycol) (PEG) polymers of different lengths, thereby gradually increasing the ligands’ range of motion in the third dimension. We found that pMHC ligands tethered by PEG linkers with long contour length were capable of activating T cells. Shorter PEG linkers, however, triggered TCR more efficiently. Molecular dynamics simulation suggested that shorter PEGs exhibit faster TCR binding on-rates and off-rates. Our findings indicate that TCR signaling can be triggered by surface-tethered pMHC ligands within a defined 3D range of motion, and that fast binding rates lead to higher TCR triggering efficiency. These observations are consistent with a model of TCR triggering that incorporates the dynamic interaction between T cell and antigen-presenting cell.     

Interaction pattern of Arg 62 in the A-pocket of differentially disease-associated HLA-B27 subtypes suggests distinct TCR binding modes

The single amino acid replacement Asp116His distinguishes the two subtypes HLA-B*2705 and HLA-B*2709 which are, respectively, associated and non-associated with Ankylosing Spondylitis, an autoimmune chronic inflammatory disease. The reason for this differential association is so far poorly understood and might be related to subtype-specific HLA:peptide conformations as well as to subtype/peptide-dependent dynamical properties on the nanoscale. Here, we combine functional experiments with extensive molecular dynamics simulations to investigate the molecular dynamics and function of the conserved Arg62 of the α1-helix for both B27 subtypes in complex with the self-peptides pVIPR (RRKWRRWHL) and TIS (RRLPIFSRL), and the viral peptides pLMP2 (RRRWRRLTV) and NPflu (SRYWAIRTR). Simulations of HLA:peptide systems suggest that peptide-stabilizing interactions of the Arg62 residue observed in crystal structures are metastable for both B27 subtypes under physiological conditions, rendering this arginine solvent-exposed and, probably, a key residue for TCR interaction more than peptide-binding. This view is supported by functional experiments with conservative (R62K) and non-conservative (R62A) B*2705 and B*2709 mutants that showed an overall reduction in their capability to present peptides to CD8+ T cells. Moreover, major subtype-dependent differences in the peptide recognition suggest distinct TCR binding modes for the B*2705 versus the B*2709 subtype.     

End-binding protein 1 controls signal propagation from the T cell receptor

The role of microtubules (MTs) in the control and dynamics of the immune synapse (IS) remains unresolved. Here, we show that T cell activation requires the growth of MTs mediated by the plus-end specific protein end-binding 1 (EB1). A direct interaction of the T cell receptor (TCR) complex with EB1 provides the molecular basis for EB1 activity promoting TCR encounter with signalling vesicles at the IS. EB1 knockdown alters TCR dynamics at the IS and prevents propagation of the TCR activation signal to LAT, thus inhibiting activation of PLCγ1 and its localization to the IS. These results identify a role for EB1 interaction with the TCR in controlling TCR sorting and its connection with the LAT/PLCγ1 signalosome.